WO2021185356A1

WO2021185356A1 - Use of butylphthalide and derivative thereof

Info

Publication number: WO2021185356A1
Application number: PCT/CN2021/081817
Authority: WO
Inventors: 刘喜宝; 李艳玲; 刘洁茹; 阿普菲尔斯图亚特; 马玉秀; 吴晓娟; 王玉青; 杨汉煜
Original assignee: 石药集团恩必普药业有限公司
Priority date: 2020-03-20
Filing date: 2021-03-19
Publication date: 2021-09-23
Also published as: US20230144023A1; EP4122462A1; JP7518183B2; AU2021239224A1; CA3175949A1; CN115243686A; JP2023522831A; KR20220156597A; AU2021239224B2; CN115243686B; EP4122462A4; CN118217279A

Abstract

The present application provides application of butylphthalide or optical isomer, prodrug, deuterium, metabolite, and ring-opening product or ring-opening product salt thereof in the preparation of a drug for preventing, alleviating, or treating peripheral neuropathy, especially chemotherapy-induced peripheral neuropathy, and a use method of butylphthalide and a derivative thereof in preventing or treating peripheral neuropathy, especially chemotherapy-induced peripheral neuropathy. In vivo and in vitro studies show that the drug of the present invention can effectively prevent, alleviate, or treat peripheral neuropathy caused by chemotherapy drugs without affecting the tumor-inhibiting efficacy and pharmacokinetic properties of the chemotherapy drugs.

Description

Use of butylphthalide and its derivatives

Cross-references to related applications

This application requires the priority and rights of the following three Chinese invention patent applications submitted to the State Intellectual Property Office of China: The application number submitted on March 20, 2020 is 202010202588.4, and the title of the invention is "Butylphthalide and its derivatives" The prior application for "use", the application number submitted on August 7, 2020 is 202010790778.2, the prior application with the title of "Usage of Butylphthalide and Its Derivatives", and the application filed on November 6, 2020 An earlier application with the number 202011233563.7 and the invention title "Uses of Butylphthalide and Its Derivatives". The full texts of the three previous applications are incorporated into this application by way of reference.

Technical field

The invention belongs to the field of medicine, and specifically relates to the application of butylphthalide and its derivatives in the preparation of drugs for preventing, relieving or treating peripheral neuropathy, especially chemotherapy-induced peripheral neuropathy, and butylphthalide and its derivatives Prevention, alleviation or treatment of peripheral neuropathy, especially chemotherapy-induced peripheral neuropathy.

Background technique

Peripheral neuropathy is a syndrome formed by loss of sensation, muscle weakness and atrophy, decreased tendon reflexes, and vascular motion symptoms, alone or in any combination. There are many peripheral neuropathies in clinic, including metabolic neuropathy, such as diabetic neuropathy; traumatic neuropathy, such as carpal tunnel syndrome; immune neuropathy, etc. Chemotherapy-induced peripheral neuropathy (chemotherapy-induced peripheral neuropathy, CIPN) is a common drug dose-limiting adverse reaction that occurs during chemotherapy in cancer patients. CIPN may be the most common type of toxic neuropathy, affecting 58% to 78% of patients receiving neurotoxic chemotherapeutics (Seretny M, Currie GL, Sena ES, et al. (2014). Incidence, prevalence, and predictors of chemotherapy-induced peripheral neuropathy: a systemic review and meta-analysis. Pain 155:2461-2470), and has serious clinical consequences. In most cases, dose-limiting toxicity limits the use of chemotherapeutic agents in the treatment of malignant tumors. The presence of toxic neuropathy usually results in a reduction in dose and sometimes the discontinuation of potentially life-saving treatments (Gadgil S, Ergun M, van den Heuval SA, et al. (2019) A systematic summary and comparison of animal models for chemotherapy induced peripheral neuropathy (CIPN). PLOS ONE https://doi.org/10.1371/journal.pone.0221787). It is estimated that among patients receiving chemotherapeutic agents that may have neurotoxicity, 30-71% of patients continue to experience CIPN-related symptoms 6 months after the start of treatment.

CIPN is mainly composed of platinum (such as cisplatin, carboplatin, oxaliplatin), paclitaxel (such as paclitaxel, docetaxel), vinblastine (such as vincristine, vinblastine), protease inhibitors (such as Bortezomib), immunomodulators (thalidomide, lenalidomide), epothilones (such as ixabepilone) and other chemotherapeutic drugs. The most common symptoms associated with CIPN are paresthesias in the hands and feet, such as: pain (burning pain, sharp pain, dullness, allodynia and hyperalgesia), tingling (paresthesia) and numbness (Ewertz M ,Qvortrup C, Eckhoff L. (2015) Chemotherapy-induced peripheral neuropathy in patients treated with taxanes and platinum derivatives. ActaOncologica 54:587-591); other common clinical manifestations include weakened or absent deep tendon reflexes, loss of body temperature or vibration , Orthostatic hypotension and sensory ataxia (especially platinum drugs); motor symptoms (such as distal or proximal weakness, muscle cramps and gait dysfunction) and autonomic symptoms (such as constipation or diarrhea, abnormal out Sweat, dizziness, and or dizziness accompanied by changes in body position) occur less frequently.

The clinical course of CIPN varies with different chemotherapy drugs. For most chemotherapy drugs, symptoms of neuropathy begin to appear after the cumulative threshold dose is reached, and continue to develop with the use of additional doses. Specific drugs, adjustment of dosing schedule, dosing frequency and even infusion speed may affect the frequency of neuropathy in a given population, but apart from stopping chemotherapy, any adjustments to the schedule will not be fully realized in the treatment population Prevent CIPN. For some chemotherapeutic agents, CIPN symptoms will continue for a period of time even if they are stopped. This phenomenon is called the "sliding phenomenon." Even if the symptoms are relatively stable, this phenomenon may continue for months, years, or indefinitely.

Two members of the taxane chemical family, paclitaxel and docetaxel (ie docetaxel) are commonly used to treat a variety of cancers. Taxane inhibits the growth of cancer by breaking down the microtubules necessary for mitotic activity, and destroys the axon transport based on microtubules through the same mechanism, and inhibits the nutritional support of sensory neurons. Taxanes are commonly used to treat breast cancer, ovarian cancer, non-small cell lung cancer, gastric cancer, head and neck cancer, and prostate cancer. Among patients receiving a cumulative dose of paclitaxel of 715-1500 mg/m² or a cumulative dose of docetaxel of 371-600 mg/m², 20-35% of patients had moderate or severe (grade 2-4) small diameters And sensory neuropathy of large fiber sensory neurons. Sensory symptoms (numbness, tingling, and pain) usually start at the distal end, first in the legs, then in the hands, which is consistent with the mechanism of action for axon transport. Among these patients, 40% of the symptoms may last for 3 years or more (Park SB, Goldstein D, Krishnan AV, et al. (2013) Chemotherapy-induced peripheral neurotoxicity: A critical analysis. CA Cancer J Clin 63: 419-437), and 50% of symptoms will recover within 9 months. Sensory neuropathy is more common and severe in patients with taxane-induced neuropathy, but some patients have decreased fine motor control ability.

Cisplatin, carboplatin, and oxaliplatin are platinum drugs used to treat cancers of the testicles, ovaries, cervix, head and neck, colon, and rectum. Their mechanism of action is to cross-link DNA and limit the ability of cancer cells to participate in DNA synthesis. When the cumulative dose is 600 mg/m 2 or more, it often leads to severe sensory ataxia after damage to large-diameter proprioceptive neurons. Fifty percent of patients who received a cumulative dose of 1170 mg/m 2 developed grade 3 sensory neuropathy related to sensory dorsal root ganglion degeneration. The most significant manifestations are tingling, numbness, loss of proprioception, and weakened or absent deep tendon reflexes, all of which indicate a large number of sensory fibers are absent in most cases (Park SB, Goldstein D, Krishnan AV, et d. (2013) Chemotherapyal. -induced peripheral neurotoxicity: A critical analysis. CA Cancer J Clin 63: 419-437), the sensory nerve conduction velocity and amplitude are also reduced. In addition to this chronic form of neuropathy, oxaliplatin is also associated with an acute type of neuropathy that occurs immediately after infusion and affects the face in addition to the limbs. The "sliding phenomenon" of platinum drugs is widespread.

Vincristine and vinblastine are the most important vinblastine drugs in the clinic, used to treat leukemia, Hodgkin's disease, malignant lymphoma, neuroblastoma, Wilms tumor, Kaposi's sarcoma, melanoma, lung cancer And uterine cancer. Their mechanism of action targets the aggregation of microtubules, causing cells to lose their ability to divide, leading to cell death. In addition to sensory deficits, 35-45% of patients treated with vincristine also have abnormal fine motor function and abnormal walking. 30% of patients will experience a "sliding phenomenon" after stopping chemotherapy (Park SB, Goldstein D, Krishnan AV, et al. (2013) Chemotherapy-induced peripheral neurotoxicity: A critical analysis. CA Cancer J Clin 63:419-437. ).

Bortezomib is a relatively new chemotherapy drug that is very effective in the treatment of multiple myeloma and other hematological malignancies. The administration of bortezomib can cause severe painful sensory neuropathy in 50% of patients, and moderate to severe neurotoxicity in about 30% of patients. Symptoms include paresthesias and numbness in the distal limbs, especially severe burning pain in the lower limbs. CIPN associated with bortezomib is small fiber sensory neuropathy. However, some patients also develop autonomic neuropathy, which may manifest as orthostatic hypotension, suppression of heart rate variability, and delayed gastric emptying. Sports injuries are rare, but they can happen sometimes. The onset dose of neuropathy is a cumulative dose of 16-26 mg/m2, and the incidence rate increases with the increase of the dose, until the dose is 40-45 mg/m2, the incidence rate tends to be stable. Neuropathy recovers slowly, and 60-85% of patients still have clinically significant neuropathy after 2-3 months of treatment.

Thalidomide and lenalidomide are synthetic glutamate derivatives used to treat relapsed and refractory multiple myeloma. CIPN is a major adverse reaction of this type of drug treatment, and the relief is slow or even irreversible. Thalidomide-induced neuropathy is mainly sensory changes, which start on the feet and extend to the hands. It is often distributed in a sock-like shape, with a heavier distal end, and does not extend to above the knees and elbows. Including hypoesthesia, hyperesthesia and dullness, muscle pain and tenderness, numbness, acupuncture sensation, burning pain, tightness, cold hands and feet, pallor, leg itching and anthurium, etc.(Bkl D, de Souza S M, Costa -Lima C,et al. Thalidomide for the treatment of gastrointestinal bleeding due to angiodysplasia in a patient with Glanzmann'sthrombasthenia[J].Hematol Rep,2017,9(2):6961). There are literatures showing that when the cumulative dose of thalidomide exceeds 20 grams, there is a positive correlation between the occurrence and severity of neuropathy and the cumulative dose (Gui Rui et al., "Medical Review" Vol 18 No 11, June 2012, P1739 -1742).

Ixabepilone (Ixabepilone) is a semi-synthetic epothilone lactam analog, it is the world's first epothilone anti-tumor drug. Compared with paclitaxel, the two drugs have different structures, but the mechanism of action is similar. Both of them interfere with the division of cancer cells to cause tumor cell apoptosis. Ixabepilone can induce peripheral lesions and make sensory nerves abnormal. This adverse reaction will generally disappear after stopping the drug for 1 to 2 months. The degree of sensory neuropathy is related to the dose and rate of administration (Burotto M, Edgerly M, Velarde M, et al. A phase II multi-center study of bevacizumab in combination with ixabepilone in subjects with advanced renal cellcarcinoma[J].Oncologist, 2017, 22(8):888-e84.)

Although CIPN is considered to be the most common serious side effect associated with chemotherapy, the clinical practice guidelines of the American Society of Clinical Oncology have concluded that there is currently no effective treatment to prevent or reverse CIPN, and only one drug duloxetine is recommended ( See Table 1 (Prevention) and Table 2 (Treatment), Hershman DL, Lacchetti C, Dworkin RH, et a. (2014).Prevention and management of chemotherapy-induced peripheral neuropathy insurvivors of adult cancers: American society of clinical practice guideline.J Clin Oncol 32:1941-67), treatment is limited to neuropathic pain drug treatment, supportive care, and chemotherapy dose adjustment (Kolb N, and Burns T (2018) Clinical Research in chemotherapy-induced peripheral neuropathy. Neurology 91:379-380). Therefore, effective prevention and treatment of CIPN is a clinical problem that needs to be solved urgently.

Table 1

Table 2

Butyl phthalide (3-n-butylphathlide, NBP), the chemical name is 3-n-butyl phthalide, also known as butyl phthalide, the trade name is Enbipu, and the structure is shown in formula (I). A drug for the treatment of mild to moderate ischemic stroke.

There is no report on the application of butylphthalide in the prevention or treatment of peripheral neuropathy, especially chemotherapy-induced peripheral neuropathy.

Summary of the invention

One of the objectives of the present invention is to provide butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or salts of ring-opening products in the preparation of drugs for the prevention, alleviation or treatment of peripheral neuropathy Applications. The peripheral neuropathy is preferably drug-induced peripheral neuropathy, and more preferably chemotherapeutic drug-induced peripheral neuropathy. The prevention, alleviation or treatment of peripheral neuropathy includes but is not limited to prevention, alleviation or treatment of paresthesias and dyskinesias caused by chemotherapeutic drugs, such as paresthesias to pain and heat, impaired motor coordination ability, and the like.

In some embodiments, the drug is in a single-dose dosage form or a divided-dose dosage form.

In some embodiments, the drug is prepared into clinically accepted preparations, such as oral preparations, injection preparations, topical administration preparations, topical preparations, etc., preferably oral preparations and injection preparations.

In some embodiments, the drug is an oral formulation. The oral preparation contains about 1 mg to about 1000 mg, preferably about 1 mg to about 500 mg, of butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product, Or about 1mg-300mg, or about 1mg-200mg, or about 5-180mg, or about 10-150mg, or about 30-120mg, or about 50-120mg, or about 80-120mg, or about 90-110mg, or about 100mg , In terms of butylphthalide.

In some embodiments, the daily dosage of the oral formulation is about 1 mg to about 10000 mg, preferably about 10 mg to about 5000 mg, or about 20 mg to 3000 mg, or about 30 mg to 2000 mg, or about 50 mg to 1500 mg, or About 70mg-1200mg, or about 100mg-1000mg, or about 200mg-900mg, or about 300mg-800mg, or about 400mg-700mg, or about 500mg-600mg, or about 60mg-800mg, or about 60mg-600mg, or about 100mg -800mg, or about 100mg-600mg, or about 200mg-600mg, or about 200mg-800mg, or about 300mg-600mg, or about 400mg-600mg, or about 400mg-800mg, in terms of butylphthalide.

In some embodiments, the oral formulation is administered once a day, and butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or salts of ring-opening products are administered each time. Oral preparations are about 60-800 mg, for example, about 60 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or 800 mg per administration of oral preparations, calculated as butylphthalide; or, twice a day, each time The oral preparation of butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product, or salt of the ring-opening product is administered about 30-400 mg, such as about 30 mg, 50 mg, 100 mg per oral preparation , 150mg, 200mg, 250mg, 300mg, 350mg or 400mg, as butylphthalide; or, administered 3 times a day, each time butylphthalide or its optical isomers, prodrugs, deuterated substances, metabolites The oral preparation of the ring-opening product or the salt of the ring-opening product is about 20-300 mg, such as about 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg or 300 mg per oral preparation, calculated as butylphthalide.

In some embodiments, the drug is an injection formulation. The injection preparation contains butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product at about 0.001mg/ml-100mg/ml, preferably, about 0.005 mg/ml-50mg/ml, or about 0.01mg/ml-10mg/ml, or about 0.1mg/ml-5mg/ml, or about 0.1mg/ml-3mg/ml, or about 0.1mg/ml-1mg/ ml, or about 0.12mg/ml-0.80mg/ml, or about 0.15mg/ml-0.50mg/ml, more preferably about 0.20mg/ml-0.40mg/ml, or about 0.20mg/ml-0.30mg/ ml, or about 0.25mg/ml, calculated as butylphthalide.

In some embodiments, the daily administration amount of the injection formulation is about 1 mg to about 1000 mg, preferably about 5 mg to about 500 mg, or about 10 mg to 300 mg, or about 15 mg to 200 mg, or about 20 mg to 150 mg, or About 25mg-120mg, or about 30mg-100mg, or about 35mg-90mg, or about 40mg-80mg, or about 45mg-70mg, or about 50mg-60mg, or about 1mg-100mg, or about 2mg-80mg, or about 5mg -75mg, or about 10mg-50mg, or about 15mg-50mg, or about 20mg-50mg, or about 25mg-75mg, or about 25mg-50mg, in terms of butylphthalide.

In some embodiments, the injection formulation is administered once a day, and butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product is administered each time. The injection preparation is about 1 mg to about 100 mg, preferably about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70 mg, 75 mg, 80 mg, 85 mg , 90mg, 95mg or 100mg, based on butylphthalide; or, twice a day, butylphthalide or its optical isomers, prodrugs, deuterated substances, metabolites, ring-opening products or The injection preparation of the salt of the ring product is about 1 mg to about 50 mg, preferably about 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg.

In some embodiments, the chemotherapeutic drugs include, but are not limited to, one or a combination of the following drugs: (1) chemotherapeutic drugs that act on the microtubule system or anti-mitosis, including: taxane drugs, such as Paclitaxel, docetaxel, etc.; or vincristine drugs, such as vincristine or vinblastine, etc.; or epothilone drugs, such as ixabepilone, etc.; or protease inhibitor drugs, such as bortezol Mi, etc.; or (2) chemotherapeutic drugs that interfere with DNA synthesis, including platinum drugs, such as cisplatin, carboplatin, or oxaliplatin; or (3) immunomodulator drugs, such as thalidomide, lenalidomide Degree amine and so on.

In some embodiments, the butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product can be combined with one or the other of other peripheral neuropathy treatment drugs. Multiple combinations are used to prepare the drug, and the other peripheral neuropathy treatment drug is preferably duloxetine or its salt, monosialotetrahexose ganglioside sodium.

Another object of the present invention also includes providing a method for preventing, alleviating or treating peripheral neuropathy, the method comprising administering to the subject or patient a therapeutically effective amount of butylphthalide or its optical isomer, prodrug, Deuterated substances, metabolites, ring-opening products or salts of ring-opening products, the peripheral neuropathy is preferably drug-induced peripheral neuropathy, and more preferably chemotherapeutic drug-induced peripheral neuropathy.

In some embodiments, the administration may be oral administration, injection administration, topical administration or in vitro administration, preferably oral administration or injection administration. The therapeutically effective amount can prevent, treat or alleviate peripheral neuropathy of the subject or patient.

In some embodiments, the administration may be oral administration. The therapeutically effective amount refers to a daily dose of about 1 mg to about 10000 mg, preferably about 10 mg to about 5000 mg, or about 20 mg to 3000 mg, or about 30 mg to 2000 mg, or about 50 mg to 1500 mg, or about 70 mg. 1200mg, or about 100mg-1000mg, or about 200mg-900mg, or about 300mg-800mg, or about 400mg-700mg, or about 500mg-600mg, or about 60mg-800mg, or about 60mg-600mg, or about 100mg-800mg, Or about 100mg-600mg, or about 200mg-600mg, or about 200mg-800mg, or about 300mg-600mg, or about 400mg-600mg, or about 400mg-800mg, in terms of butylphthalide.

In some embodiments, the administration may be injection administration. The therapeutically effective amount refers to a daily dose of about 1 mg to about 1000 mg, preferably about 5 mg to about 500 mg, or about 10 mg to 300 mg, or about 15 mg to 200 mg, or about 20 mg to 150 mg, or about 25 mg. 120mg, or about 30mg-100mg, or about 35mg-90mg, or about 40mg-80mg, or about 45mg-70mg, or about 50mg-60mg, or about 1mg-100mg, or about 2mg-80mg, or about 5mg-75mg, Or about 10mg-50mg, or about 15mg-50mg, or about 20mg-50mg, or about 25mg-75mg, or about 25mg-50mg, based on butylphthalide.

In some embodiments, butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product, or salt of the ring-opening product is administered in a single dose or in divided doses.

In some embodiments, the prevention, alleviation or treatment method is: once a day, butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or open The oral preparation of the salt of the cyclic product is about 60-800 mg, for example, about 60 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg or 800 mg of the oral preparation per administration, calculated as butylphthalide; or daily administration 2 times, about 30-400 mg of oral preparations of butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or salts of ring-opening products are administered each time, such as about 30-400 mg each time the oral preparation is administered 30mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg or 400mg, calculated as butylphthalide; or, administered 3 times a day, each time butylphthalide or its optical isomers, prodrugs, Oral preparations of deuterated substances, metabolites, ring-opening products or their salts are about 20-300 mg, such as about 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg or 300 mg of oral preparations per administration, calculated as butylphthalide; or, It is administered once a day, and the injection preparation of butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product is administered at about 1 mg to about 100 mg each time, preferably, For about 1mg, 2mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg or 100mg, with butylbenzene Phthalide meter; or, twice a day, about 1 mg-about injection preparation of butylphthalide or its optical isomers, prodrugs, deuterated substances, metabolites, ring-opening products or salts of ring-opening products are administered each time 50 mg, preferably, is about 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, in terms of butylphthalide.

In some embodiments, the butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product can be combined with one or the other of other peripheral neuropathy treatment drugs. Multiple combinations are used, and the other therapeutic drugs for peripheral neuropathy are preferably duloxetine or its salt, monosialotetrahexose ganglioside sodium.

Another object of the present invention also includes providing a kind of butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product, which is used to prevent, alleviate or treat surrounding Neuropathy. The peripheral neuropathy is preferably drug-induced peripheral neuropathy, and more preferably chemotherapeutic drug-induced peripheral neuropathy.

In some embodiments, the prevention, alleviation or treatment includes administering to the subject or patient a therapeutically effective amount of butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or ring-opening The salt of the product.

In some embodiments, the administration is selected from oral administration, injection administration, topical administration or in vitro administration, preferably oral administration or injection administration. The therapeutically effective amount can prevent, alleviate or treat peripheral neuropathy of the subject or patient.

In some embodiments, the administration is oral administration. The therapeutically effective amount refers to a daily dose of about 1 mg to about 10000 mg, preferably about 10 mg to about 5000 mg, or about 20 mg to 3000 mg, or about 30 mg to 2000 mg, or about 50 mg to 1500 mg, or about 70 mg. 1200mg, or about 100mg-1000mg, or about 200mg-900mg, or about 300mg-800mg, or about 400mg-700mg, or about 500mg-600mg, or about 60mg-800mg, or about 60mg-600mg, or about 100mg-800mg, Or about 100mg-600mg, or about 200mg-600mg, or about 200mg-800mg, or about 300mg-600mg, or about 400mg-600mg, or about 400mg-800mg, in terms of butylphthalide.

In some embodiments, the administration is by injection. The therapeutically effective amount refers to a daily dose of about 1 mg to about 1000 mg, preferably about 5 mg to about 500 mg, or about 10 mg to 300 mg, or about 15 mg to 200 mg, or about 20 mg to 150 mg, or about 25 mg. 120mg, or about 30mg-100mg, or about 35mg-90mg, or about 40mg-80mg, or about 45mg-70mg, or about 50mg-60mg, or about 1mg-100mg, or about 2mg-80mg, or about 5mg-75mg, Or about 10mg-50mg, or about 15mg-50mg, or about 20mg-50mg, or about 25mg-75mg, or about 25mg-50mg, based on butylphthalide.

In some embodiments, the prevention, alleviation or treatment method is: once a day, butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or open The oral preparation of the salt of the cyclic product is about 60-800 mg, for example, about 60 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or 800 mg of the oral preparation per administration, in terms of butylphthalide; or daily administration 2 times, about 30-400 mg of oral preparations of butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or salts of ring-opening products are administered each time, such as about 30-400 mg each time the oral preparation is administered 30mg, 100mg, 200mg, 300mg or 400mg, calculated as butylphthalide; or, administered 3 times a day, butylphthalide or its optical isomers, prodrugs, deuterated substances, metabolites, ring opening The oral preparation of the salt of the product or the ring-opening product is about 20-300 mg, such as about 20 mg, 100 mg, 200 mg, or 300 mg per administration of the oral preparation, calculated as butylphthalide; or, once a day, butyl is administered each time The injection preparation of phthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product is about 1 mg to about 100 mg, preferably about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg , 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, 55mg, 60mg, 65mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg or 100mg, in terms of butylphthalide; or, twice a day, Each injection of butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product is administered at about 1 mg to about 50 mg, preferably about 1 mg, 2 mg, 2.5mg, 5mg, 10mg, 15mg, 20mg, 25mg, 30mg, 35mg, 40mg, 45mg, 50mg, in terms of butylphthalide.

In some embodiments, the chemotherapeutic drugs include, but are not limited to, one or more of the following drugs: (1) chemotherapeutic drugs that act on the microtubule system or anti-mitosis, including: taxane drugs, such as paclitaxel, Docetaxel, etc.; or vincristine drugs, such as vincristine or vinblastine, etc.; or epothilone drugs, such as ixabepilone, etc.; or protease inhibitor drugs, such as bortezomib, etc. ; Or (2) Chemotherapy drugs that interfere with DNA synthesis, including platinum drugs, such as cisplatin, carboplatin, or oxaliplatin; or (3) Immunomodulator drugs, such as thalidomide and lenalidomide Wait.

Another object of the present invention also includes providing a pharmaceutical composition containing butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or salts of ring-opening products, the drug The composition is used to prevent, relieve or treat peripheral neuropathy. The peripheral neuropathy is preferably drug-induced peripheral neuropathy, and more preferably chemotherapeutic drug-induced peripheral neuropathy.

The optical isomers of butylphthalide in the present invention are L-butylphthalide, dextrobutylphthalide, or a mixture of the two in any ratio.

The prodrugs of butylphthalide in the present invention refer to compounds designed according to the principle of prodrugs. Such compounds can be metabolized into butylphthalide in the body and exert the same or similar pharmacological effects as butylphthalide.

The deuterated product of butylphthalide in the present invention means that one or several hydrogen atoms in the structure of the p-butylphthalide compound are replaced by its isotope atom deuterium. This kind of deuterated product has the same or Similar pharmacological effects. Deuterated derivatives of butylphthalide and its derivatives as described in WO2019242765A1, especially deuterated derivatives of butylphthalide with the following structure:

The metabolites of butylphthalide in the present invention refer to the in vivo metabolites of butylphthalide or derivatives thereof, and such metabolites can exert the same or similar pharmacological effects as butylphthalide. Such as the butylphthalide metabolites MI and MII described in CN1689563A, and the ester or salt of MI described in CN102503919A or CN101289438A:

The ring-opening product or salt of the ring-opening product of butylphthalide in the present invention refers to the product or salt thereof obtained by ring-opening of the lactone in the butylphthalide structure. Such ring-opening products can be combined to form a ring in the body Butylphthalide exerts the same or similar pharmacological effects as butylphthalide. Ring-opening products or salts of butylphthalide as described in CN1382682A, CN1523003A and CN104974050A:

Wherein, M is a monovalent metal ion, which is potassium ion, sodium ion or lithium ion; or a divalent metal ion, which is calcium ion, magnesium ion or zinc ion, wherein n=1, or n=2; M is preferably Is potassium ion;

And its optical isomers;

And the glycosyl-modified butylphthalide ring-opening derivatives described in CN108084233A, the butylphthalide ring-opening derivatives described in CN109503548A, the organic amino ester derivatives of 2-(αhydroxypentyl)benzoic acid described in CN108715579A, etc. .

The dosage of the butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or salts of the ring-opening products of the present invention are all based on butylphthalide.

Those skilled in the art can understand that other butylphthalide analogs or their optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or salts of ring-opening products, such as 7-hydroxybutylphthalide described in CN106214674A , Halobutylphthalide described in CN101029037A (especially 5-bromobutylphthalide), halogenated 2-(α hydroxypentyl) benzoate described in CN101402565A, 5-bromo-2 described in CN104086399A -(α-hydroxypentyl) benzoic acid sodium salt, etc., will also exert a pharmacological effect similar to that of butylphthalide.

In the technical solution of this application, the "about" before the value refers to ±10% of the value, preferably ±5%, for example, ±10%, ±9%, ±8%, ±7%, ±10% of the value. 6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

Technical effect

1. The representative drug butylphthalide of the present invention has a significant improvement effect on peripheral neuropathy induced by paclitaxel or bortezomib, and can obviously reverse the symptoms of abnormal thermal pain threshold and mechanical pain threshold caused by these two chemotherapeutic drugs.

2. Butylphthalide, the representative drug of the present invention, does not weaken the activity of bortezomib and paclitaxel in inhibiting the proliferation of tumor cells cultured in vitro, and has no significant effect on the in vivo tumor suppressive effect and pharmacokinetic properties of bortezomib and paclitaxel. .

3. In vivo and in vitro experiments show that the drug of the present invention can treat or relieve peripheral neuropathy caused by chemotherapy drugs without affecting the anti-tumor efficacy and pharmacokinetic properties of the chemotherapy drugs.

Description of the drawings

FIG. 1 is a schematic diagram of the method of making a model in Example 1. FIG.

Figure 2 is a graph showing changes in body weight of rats in each group from day 0 to day 16 (D0 to 16) in Example 1. Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Fig. 3 is a graph showing changes in heat pain threshold of rats in each group from day 0 to day 13 (D0 to 13) in Example 1. Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 4 is a comparison of the thermal pain thresholds of rats in each group on 7/10/13 days (D7/D10/D13) in Example 1. Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 5 is the timeline of model building and drug administration in Example 2.

Fig. 6 is a graph showing the change in body weight of rats in Example 2 (mean±SD, n=12). Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Fig. 7 is the change of the heat pain threshold of Example 2 rats on day -1/7/14 (mean ± SD, n=12). Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Fig. 8 shows the changes in mechanical pain threshold of Example 2 rats on day 0/6/13 (mean±SD, n=12). Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 9 shows the changes in body weight of rats in each group in Example 3. Compared with the solvent group, *P≤0.05, **P≤0.01, ***P≤0.001; compared with the ab-PTX group, ##P≤0.01.

Figure 10 shows the changes in body weight of rats in each group in Example 4. After statistical analysis, compared with the solvent group, *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 11 is the timeline of model building and drug administration in Example 6.

Figure 12 shows the changes in body weight of rats in each group from D0 to 20 in Example 6. Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 13 shows the changes in the thermal pain threshold of rats in Example 6. Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Figure 14 shows the changes in the mechanical pain threshold of rats in Example 6. Compared with the model group, *P≤0.05, **P≤0.01, ***P≤0.001.

Fig. 15 is a graph showing the change in body weight of mice in each group in Example 8 (mean±SD, n=6).

Figure 16 is a graph showing the change of tumor volume in tumor-bearing mice in Example 8 (mean ± SD, n = 6; compared with the solvent group, ^* P≤0.05, ^** P≤0.01, ^*** P≤0.001 ).

Note: In the figure, mpk is equivalent to mg/kg.

Detailed ways

The examples listed below are to better illustrate the content of the present invention, but the content of the present invention is not limited to the examples. Those skilled in the art make non-essential improvements and adjustments to the embodiments based on the above-mentioned content of the invention, which still belongs to the protection scope of the present invention.

Example 1: In vivo pharmacodynamic study of butylphthalide (intraperitoneal injection) on paclitaxel (albumin-binding) chemotherapy-induced peripheral neuropathy in rats

1. Experimental animals

SD (Sprague Dawley) rats, male, 180-200g, 59 rats.

2. Drugs

Paclitaxel (Albumin Bound) (Paclitaxel (Albumin Bound), ab-PTX) freeze-dried powder, 100mg/bottle, made by CSPC Ouyi Pharmaceutical Co., Ltd.; batch number: B041909121, dissolved in physiological saline before use.

Butylphthalide (NBP) injection, 25mg/5mL, made by CSPC Enbipu Pharmaceutical Co., Ltd.; batch number: Q27190401, diluted with normal saline to the target concentration before use.

3. Test method

(1) Model preparation and administration method

Male SD rats, ab-PTX 5mg/kg, administration volume of 5mL/kg, intraperitoneal injection (ip) on

days

0, 2, 4, 6, 8, and 11 were used to create the model; NBP used 1.5, 5, and Three doses of 15mg/kg bid, started 1 day before the ab-PTX model, during the ab-PTX administration model, the first administration of NBP was given by intraperitoneal injection 1 hour before the ab-PTX administration, NBP The interval between the second administration and the first administration was 4h, the administration volume was 5mL/kg, and the administration was given every day for 17 consecutive days; the normal group (Normal) was given 0.9% saline (0.9% INJ NS) at the same frequency . The modeling method is shown in Figure 1, and the animal grouping, administration method and dosage are shown in Table 3.

Table 3 Example 1 Animal Grouping and Dosage Table

Note: (1) bid: twice a day; (2) three doses of NBP, converted into adult daily dose (calculated by 60kg, orally) are 60mg/d, 200mg/d, 600mg/d.

(2) Group

According to the baseline value of the heat pain threshold and body weight of the rats, the rats with the baseline value in the range of 3-6s were selected, and the rats were divided into groups according to the above-mentioned Table 3.

(3) Observation indicators

①Body weight: All animals were weighed once before the test, and weighed at a fixed time every day after the start of the administration, but only the statistics on the -1, 0, 2, 4, 6, 8, 10, 12, 14 and 16 days Weight data.

②Thermal pain threshold (thermal pain threshold): Using the IITC Life Science Electronic Von Frey Anesthesio meter, adjust the light source intensity to obtain the thermal pain threshold baseline for about 3 to 6 seconds. At this time, the light intensity is 58%, and Set the cutoff value to 10s. Place the rat in the plastic compartment on the glass plate, and turn on the light source to illuminate the center of the rat's foot. Record the time of reflex withdrawal of the rat, that is, the thermal pain threshold. The measurement is performed once for each rat's left and right feet, and the measurement is repeated three times with an interval of ≥15 min. The average of the 6 thermal pain thresholds for each rat is calculated.

4. Experimental results

(1) Weight

On days 4 to 16 (D4-16), compared with the normal group, the weight of rats in the model group was significantly reduced (P<0.01); compared with the model group, NBP 1.5, 5, and 15 mg/kg bid The body weight of rats in the dose group did not change significantly, as shown in Figure 2.

(2) Thermal pain threshold

The measurement was performed on the 7, 10, 13 and 17 days. On days 7 to 13 (D7-13), compared with the normal group, the thermal pain threshold of the model group was significantly increased (P<0.01); compared with the model group, the NBP 1.5, 5, and 10 mg/kg bid groups were all significantly higher Reduce the increase in thermal pain threshold of model rats (P<0.05 or P<0.01); see Figure 3 and Figure 4 for details.

On day 17 (D17), compared with the normal group, there was no statistical difference in the heat pain threshold of the model group (D17 data is not shown). Therefore, the heat pain threshold of the NBP administration group was not detected and the experiment was terminated.

5 Conclusion

Butylphthalide can effectively alleviate the abnormal symptoms of heat pain in rats with peripheral neuropathy induced by paclitaxel (albumin-bound type).

Example 2: In vivo pharmacodynamic study of butylphthalide (administered by intragastric administration) on the peripheral neuropathy of rats induced by chemotherapy of paclitaxel (albumin-binding type)

1. Experimental animals

SD (Sprague Dawley) rats, male, 160-180g, 72 rats.

2. Drugs

Paclitaxel (albumin-bound, ab-PTX) freeze-dried powder, 100mg/bottle, batch number: B041909121, made by CSPC Ouyi Pharmaceutical Co., Ltd., dissolved in normal saline before use.

Butylphthalide, oral grade (10kg/bottle), batch number: 518180803, made by CSPC Enbipu Pharmaceutical Co., Ltd., diluted with vegetable oil to the target concentration before use.

Duloxetine hydrochloride, batch number: QRQYD-JN, Tixiai (Shanghai) Chemical Industry Development Co., Ltd., prepared with 2% DMSO in physiological saline before use.

3. Test method

3.1 Model preparation and administration method

This test uses male SD rats, ab-PTX 5mg/kg, the dose volume is 5mL/kg, on the 0, 2, 4, 6, 9, and 13 days (the first dose of ab-PTX is the test 0 Day) Intraperitoneal injection (ip) for modeling.

NBP uses three doses of 3, 10, and 30 mg/kg bid, and the positive control drug duloxetine hydrochloride 15 mg/kg qd. Both NBP and duloxetine hydrochloride were administered 1 day before the ab-PTX model was established. During the ab-PTX administration model, both NBP (first administration) and duloxetine hydrochloride were injected intraperitoneally in the ab-PTX 1h before administration, the interval between the second administration of NBP and the first administration is ≥4h, the administration volume is 5mL/kg, and the intragastric administration is given for 14 consecutive days.

The model group was given vegetable oil at the same frequency and administration method as NBP.

The normal group was given physiological saline at the same frequency and administration method as ab-PTX, and vegetable oil was given at the same frequency and administration method as NBP.

The modeling method and administration time are shown in Fig. 5, and the animal grouping, administration method and dosage are shown in Table 4.

Table 4 Animal grouping and dosage table

Group

Number of animals (only)

drug

Dose (mg/kg)

Route of administration

正常组 normal group	1212	植物油/生理盐水Vegetable oil/physiological saline	------	p.o./i.p.p.o./i.p.
模型组 Model group	1212	植物油/ab-PTXVegetable oil/ab-PTX	55	p.o./i.p.p.o./i.p.
NBP3mg/kg bidNBP3mg/kg bid	1212	NBP/ab-PTXNBP/ab-PTX	3/53/5	p.o./i.p.p.o./i.p.
NBP10mg/kg bidNBP10mg/kg bid	1212	NBP/ab-PTXNBP/ab-PTX	10/510/5	p.o./i.p.p.o./i.p.
NBP30mg/kg bidNBP30mg/kg bid	1212	NBP/ab-PTXNBP/ab-PTX	30/530/5	p.o./i.p.p.o./i.p.
盐酸度洛西汀15mg/kg qdDuloxetine hydrochloride 15mg/kg qd	1212	盐酸度洛西汀/ab-PTXDuloxetine Hydrochloride/ab-PTX	15/515/5	p.o./i.p.p.o./i.p.

Note: (1) po: gavage; (2) ip: intraperitoneal injection; (3) three doses of NBP, converted to adult daily dose (calculated as 60kg, orally) are 60mg/d and 200mg/ d, 600mg/d.

3.2 Grouping

According to the baseline value of the heat pain threshold and body weight of the rats, the rats with the baseline value in the range of 3 to 6 s were selected, and the rats were divided into groups according to 3.1 items in Table 4 below.

3.3 Observation indicators

(1) Body weight: All animals were weighed once before the test, and weighed at a fixed time every day after the start of dosing, but only statistics on days -1, 0, 2, 4, 6, 8, 10, 12, and 14 Weight data.

(2) Thermal pain threshold: measured on the -1, 7, and 14 days.

Method: Using the electronic plantar pain meter (IITC Life Science), adjust the intensity of the light source to obtain a baseline of the thermal pain threshold of about 3 to 6 seconds. At this time, the light intensity is 58%, and the cut-off value is set to 10s. Place the rat in the plastic compartment on the glass plate, and turn on the light source to illuminate the center of the rat's foot. Record the time of reflex withdrawal of the rat, that is, the thermal pain threshold. The measurement is performed once for each rat's left and right feet, and the measurement is repeated three times with an interval of ≥15 min. The average of the 6 thermal pain thresholds for each rat is calculated.

(3) Mechanical stimulation pain threshold: measured on the 0th, 6th, and 13th days.

Method: Using an electronic mechanical stinging instrument (IITC Life Science), the rat was placed in a plastic compartment on a barbed wire and allowed to stand for 15 minutes. A stimulus probe with a diameter of 0.4 mm was selected and aimed at the center of the rat’s foot, from small to large. Gradually increase the force until the rat reflexively withdraws, and record the maximum value displayed by the instrument at this time, that is, the mechanical pain threshold. The left and right feet of each rat are measured three times for each test, each time interval is ≥5min, and each rat is calculated as 6 Average value of secondary mechanical pain threshold.

4. Results

4.1 Impact on weight

On days 4 to 14 of the test, compared with the normal group, the weight of rats in the model group was significantly reduced (P<0.05); during the entire administration cycle, compared with the model group, three doses of

NBP

3, 10 and 30 mg/kg bid The body weight of the rats in the group did not change significantly, and the positive drug duloxetine hydrochloride could significantly increase the body weight of the model rats (P<0.05); see Figure 6 for details.

4.2 Impact on thermal pain threshold

On the 7th day of the test, compared with the normal group, the heat pain threshold of rats in the model group was significantly increased (P<0.01); compared with the model group, the heat pain threshold of rats in the NBP 30mg/kg bid group was significantly lower (P<0.05) ).

On the 14th day of the test, compared with the normal group, the heat pain threshold of the model group was significantly increased (P<0.001); compared with the model group, the three dose groups of

NBP

3, 10 and 30 mg/kg bid, positive drug salt The heat pain threshold of 15 mg/kg acid duloxetine qd rats was significantly reduced (P<0.05), see Figure 7 for details.

4.3 Impact on mechanical pain threshold

On the 6th day of the test, compared with the normal group, the mechanical pain threshold of rats in the model group was significantly increased (P<0.05); compared with the model group, the mechanical pain threshold of rats in the NBP 30mg/kg bid group was significantly lower (P<0.05) ).

On the 13th day of the test, compared with the normal group, the mechanical pain threshold of the model group was significantly increased (P<0.001); compared with the model group, the

NBP

10 and 30 mg/kg bid dose groups, the positive drug duloxetine hydrochloride The mechanical pain threshold of rats at 15 mg/kg qd decreased significantly (P<0.05). The mechanical pain threshold of rats in the NBP 3 mg/kg bid dose group had a decreasing trend, but there was no statistical difference; see Figure 8 for details.

6 Conclusion

Under the conditions of this experiment, NBP can effectively relieve the symptoms of peripheral neuropathy in the rat PIPN (Paclitaxel-Induced Peripheral Neuropathy, PIPN) model induced by ab-PTX in a dose-dependent manner.

Example 3 The effect of NBP on the anti-tumor efficacy and PK of ab-PTX against B16/F10 xenografts

1. Test system

1.1 Experimental animals

C57BL/6N mice, female, 15-17g, 40 mice.

1.2 cell line

B16/F10 cells (mouse melanoma cells): purchased from Shanghai Jikai Gene Chemical Technology Co., Ltd.

2. Drugs

Paclitaxel (albumin-bound type) freeze-dried powder, 100mg/bottle, batch number: B041909121, made by CSPC Ouyi Pharmaceutical Co., Ltd., dissolved in normal saline before use.

Butylphthalide, 25mg/5mL/bottle, batch number: Q27190401, made by CSPC Enbipu Pharmaceutical Co., Ltd., dilute with normal saline to the target concentration before use.

3. Test method

3.1 Model preparation

After resuscitating and passage B16/F10 cells in vitro to a sufficient amount, after counting by microscope, the cells were diluted with serum-free medium to adjust the cell number to about 1×10 ⁷ cells/mL, and the cell suspension was placed in an ice water bath.

The B16/F10 cell suspension was drawn with a sterile syringe and inoculated into the subcutaneous tissue of the forelimb axilla of C57BL/6N mice. The inoculation volume was 0.1 mL/head, containing about 1.0×10 ⁶ tumor cells to prepare C57BL/6N mice B16/F10 Transplanted tumor model.

3.2 Method of administration

G:

Ab-PTX single-drug group: ab-PTX 25mg/kg, biw was given by intraperitoneal injection twice a week (on the 1, 4, 8, 11, and 15 days, once a day). The administration volume is 10 mL/kg.

NBP/ab-PTX group: give ab-PTX 25mg/kg, biw by intraperitoneal injection twice a week (on the 1, 4, 8, 11, and 15 days, once a day); give NBP 3mg/kg by intraperitoneal injection twice a day , 10mg/kg or 30mg/kg, bid (administration started on day 0). During the ab-PTX modeling period, the first administration of NBP was given by intraperitoneal injection 1h before the administration of ab-PTX, and the second administration was with The interval between the first dosing is greater than 4h. The administration was continued for 15 days. The administration volume is 10mL/kg.

Solvent group: Inject solvent (normal saline) into the abdominal cavity at the same frequency and administration volume as ab-PTX and NBP.

Note: The three doses of NBP, converted to adult daily dose (calculated as 60kg, orally) are 60mg/d, 200mg/d, and 600mg/d respectively.

3.3 Observation indicators and evaluation indicators

3.3.1 General observation indicators

(1) Observation of general status: All animals were observed once a day during the test period, and abnormalities in various parts of the body and changes in behavior were recorded.

(2) Body weight: All animals were weighed once before the test, and animals with a suitable weight were selected for the test. After the start of administration, the animals were weighed once a day at a fixed time.

(3) Death and dying: record the time of death of dead animals, and increase the frequency of observation for dying animals to determine the time of death.

3.3.2 Tumor weight

At the end of the experiment, after the animal was _{euthanized by CO 2} asphyxiation, the tumor was stripped and weighed.

Tumor weight inhibition rate% = (1-Tumor weight in the administration group/tumor weight in the solvent group) × 100%

3.3.3 PK blood sampling and testing

Before the last administration of ab-PTX (0h), 5min, 0.5h, 1h, 4h, 8h, 12h, 24h after the administration, blood was collected (see Table 5 for details), heparin was anticoagulated, the plasma was separated by centrifugation, and stored at -80°C for later use. The total amount of paclitaxel in plasma was determined by protein precipitation-LC/MS/MS method.

Table 5 Blood collection schedule of mice in each group

组别Group	动物编号Animal number	采血时间点Blood sampling time point

溶剂组Solvent group	1～81～8	0h0h
各给药组Each administration group	1～41～4	0h，0.5h，8h，12h0h, 0.5h, 8h, 12h
各给药组Each administration group	5～85～8	5min，1h，8h，24h5min, 1h, 8h, 24h

4 results

4.1 Impact on weight

Compared with the solvent group, the weight gain rate of mice in each administration group was significantly reduced (P<0.01). Compared with the ab-PTX group, the weight gain rate of the NBP/ab-PTX 10/25mg/kg dose group was significantly reduced (P＜0.01). Combined with the analysis of tumor weight data, the reduction in weight gain rate of each administration group should be due to drug inhibition Heavy results. See Figure 9 for details.

4.2 Impact on tumor weight

Compared with the solvent group, each administration group can significantly inhibit tumor growth (P<0.001); compared with the ab-PTX group, the combined administration of different doses of NBP and ab-PTX has no statistical difference in tumor inhibition ( P>0.05). The tumor weight of each group is shown in Table 6.

Table 6 Effect on tumor weight in mice (mean±SD, n=8)

组别Group	动物数(存活/总)Number of animals (survival/total)	瘤重(g)Tumor weight (g)	抑制率(％)Inhibition rate(%)
溶剂组 Solvent group	8/88/8	5.2178±1.51465.2178±1.5146	------
NBP/ab-PTX 3/25mg/kgNBP/ab-PTX 3/25mg/kg	8/88/8	1.9635±0.7882 ^* 1.9635±0.7882 ^*	62.462.4
NBP/ab-PTX 10/25mg/kgNBP/ab-PTX 10/25mg/kg	8/88/8	1.7374±0.5333 ^* 1.7374±0.5333 ^*	66.766.7
NBP/ab-PTX 30/25mg/kgNBP/ab-PTX 30/25mg/kg	8/88/8	1.8226±0.9578 ^* 1.8226±0.9578 ^*	65.165.1
ab-PTX 25mg/kgab-PTX 25mg/kg	8/88/8	2.5024±0.8133 ^* 2.5024±0.8133 ^*	52.052.0

Note: Compared with the solvent group: ***P＜0.001.

4.3 Impact on PK

The protein precipitation-LC/MS/MS method was used to determine the total amount of paclitaxel in plasma. The results showed that _{there were no significant statistical differences in C max} and AUC _0-t among the administration groups, indicating that NBP did not affect the ab-PTX in PK behavior in mice. See Table 7 for details.

Table 7 Influence on pharmacokinetic parameters

5 Conclusion

In this experiment, NBP had no significant effect on the anti-tumor efficacy of ab-PTX and the PK behavior of ab-PTX.

Example 4 The effect of NBP on the anti-tumor efficacy and PK of ab-PTX against JIMT-1 transplanted tumor

1. Test system

1.1 Experimental animals

Nu/Nu mice, female, 15-17g, 48 mice.

1.2 cell line

JIMT-1 cells (human breast cancer cells): purchased from Nanjing Kebai Biotechnology Co., Ltd.

2. Test purpose

In this experiment, a Nu/Nu mouse JIMT-1 cell transplantation tumor model was used to verify the effect of NBP on the anti-tumor efficacy and PK behavior of ab-PTX.

3. Drugs

Paclitaxel (albumin-bound type) freeze-dried powder, 100mg/bottle, batch number: B042007410, made by CSPC Ouyi Pharmaceutical Co., Ltd., dissolved in normal saline before use.

4. Test method

4.1 Model preparation

After resuscitating and passage JIMT-1 cells in vitro to a sufficient amount, after counting by microscope, dilute the cells with serum-free medium to adjust the number of cells to about 1×10 ⁸ cells/mL, and place the cell suspension in an ice water bath.

The JIMT-1 cell suspension was drawn with a sterile syringe and inoculated into the subcutaneous tissue of the forelimb axillary of Nu/Nu mice. The inoculation volume was 0.1 mL/head, containing about 1.0×10 ⁷ tumor cells to prepare Nu/Nu mouse JIMT-1 Transplanted tumor model.

4.2 Method of administration

Each administration group:

Ab-PTX single-drug group: give ab-PTX 15 mg/kg, qw intravenously once a week (

day

0, 7, 14, 21, 28). The administration cycle is 28 days. The administration volume is 10 mL/kg.

NBP single-drug group: give NBP 60 mg/kg by gavage twice a day, bid (administration started on day 0), the interval of administration must be greater than 4 hours; the administration cycle is 28 days. The administration volume is 10mL/kg.

NBP/ab-PTX group: give ab-PTX 15 mg/kg, qw intravenously once a week (

days

0, 7, 14, 21, 28). Orally give NBP 6mg/kg, 20mg/kg or 60mg/kg twice a day, bid (administration starting on day 0), the interval of administration should be greater than 4h; the first administration of NBP is 1h before the ab-PTX administration . The administration cycle is 28 days. The administration volume is 10mL/kg.

The solvent group was given physiological saline at the same frequency and method as ab-PTX, and vegetable oil was given at the same frequency and method as NBP.

Note: The dosage of NBP is 60mg/d, 200mg/d, and 600mg/d.

4.3 Observation indicators and evaluation indicators

4.3.1 General observation indicators

4.3.2 Tumor weight

Tumor weight inhibition rate% = (1-tumor weight in the administration group/tumor weight in the solvent group) × 100%.

4.3.3 PK blood sampling and testing

Before the last administration of ab-PTX (0h), 5min, 15min, 0.5h, 1h, 4h, 8h, 24h after the administration (see Table 8 for details), heparin anticoagulation, centrifugal separation of plasma, and storage at -80℃ for later use. The total amount of paclitaxel in plasma was determined by protein precipitation-LC/MS/MS method.

Table 8 Blood collection schedule of mice in each group

组别Group	动物编号Animal number	采血时间点Blood sampling time point

溶剂组Solvent group	1～81～8	0h0h
各给药组Each administration group	1～81～8	0h，5min，15min，0.5h，1h，4h，8h，24h0h, 5min, 15min, 0.5h, 1h, 4h, 8h, 24h

5. Results

5.1 Impact on weight

From the body weight data, there was no significant difference in the body weight of the mice in each group, indicating that neither ab-PTX nor NBP had a significant effect on the body weight of the mice. See Figure 10 for details.

5.2 Impact on tumor weight

The tumor weight results showed that compared with the solvent group, the ab-PTX administration group could significantly inhibit tumor growth (P＜0.001), and the NBP 60mg/kg dose group had no significant effect on mouse tumor growth; compared with ab-PTX 15mg/kg Compared with the group, the combined administration of NBP and ab-PTX at different doses had no statistically significant difference in tumor inhibition (P>0.05). See Table 9 for details.

Table 9 Effect on tumor weight in mice (mean±SD, n=8)

组别Group	动物数(存活/总)Number of animals (survival/total)	瘤重(g)Tumor weight (g)	抑制率(％)Inhibition rate(%)
溶剂组 Solvent group	8/88/8	0.9969±0.24260.9969±0.2426	------
ab-PTX15mg/kgab-PTX15mg/kg	8/88/8	0.2789±0.0858*0.2789±0.0858*	72.072.0
NBP/ab-PTX6/15mg/kgNBP/ab-PTX6/15mg/kg	8/88/8	0.2466±0.1651*0.2466±0.1651*	75.375.3
NBP/ab-PTX20/15mg/kgNBP/ab-PTX20/15mg/kg	8/88/8	0.2807±0.1309*0.2807±0.1309*	71.871.8

NBP/ab-PTX60/15mg/kgNBP/ab-PTX60/15mg/kg	8/88/8	0.3140±0.1724*0.3140±0.1724*	68.568.5
NBP60mg/kgNBP60mg/kg	8/88/8	0.9570±0.25290.9570±0.2529	4.04.0

Note: Compared with the solvent group: ***P＜0.001.

5.4 Impact on PK

The results showed that the main pharmacokinetic parameters of paclitaxel in the plasma of the ab-PTX single-agent group and the NBP and ab-PTX combined administration group were basically the same, and there was no significant difference. It shows that the combined administration of butylphthalide and ab-PTX does not affect the pharmacokinetic behavior of paclitaxel in plasma. See Table 10 for details.

Table 10 Main pharmacokinetic parameters of total paclitaxel in each administration group

6 Conclusion

In this trial, NBP had no significant effect on the anti-tumor efficacy of ab-PTX and the PK behavior of ab-PTX.

Example 5 The effect of NBP on the in vitro efficacy of ab-PTX

1. Cells and culture environment

Table 11 Cells and required medium

The above cell culture environment is 37°C and 5% CO ₂ .

2. Test purpose

To investigate whether NBP affects the inhibitory effect of ab-PTX on tumor cell proliferation in vitro.

3. Drugs

Paclitaxel (albumin-bound type) freeze-dried powder, 100mg/bottle, batch number: B041909121, made by CSPC Ouyi Pharmaceutical Co., Ltd.

Butylphthalide, oral grade (10kg/bottle), batch number: 518180803, made by CSPC Enbipu Pharmaceutical Co., Ltd.

4. Experimental design

The test is divided into ab-PTX single-drug group, combined administration group (ab-PTX/NBP), and NBP group. Each group has a different drug concentration gradient.

ab-PTX single-drug group: ① MDA-MB-231, JIMT-1, SK-OV-3, A549 cell lines: ab-PTX starts with 200 nM, and 3×decreasing dilution sets a total of 8 concentrations, each of which is 200 , 66.67, 22.22, 7.41, 2.47, 0.82, 0.27, 0.09nM. ②A375 and HT29 cell lines: the initial test of ab-PTX starts with 100nM, and 3×decreasing dilution sets a total of 8 concentrations, which are 100, 33.33, 11.11, 3.70, 1.24, 0.41, 0.14, 0.05nM; in repeated experiments The ab-PTX starts with 100nM, and 2×decreasing dilution sets a total of 8 concentrations, namely 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78nM.

ab-PTX/NBP group: The final concentration of NBP is 30μM, and the concentration gradient of ab-PTX is the same as that of the single-drug group.

NBP single-drug group: NBP is 480μM as the starting concentration, 2×decreasing dilution is set to 8 concentrations, respectively 480, 240, 120, 60, 30, 15, 7.5, 3.75μM.

The action time of the drug was 72h.

5. Test method

Cells in the logarithmic growth phase of conventional culture were seeded in a 96-well plate in a certain amount, with 100 μL per well. After 24 hours of adherence, the ab-PTX single-drug group added 100 μL of culture medium containing different concentration gradients of ab-PTX to each well; the ab-PTX/NBP combined administration group added 100 μL of different concentration gradients of ab-PTX and 30 μM NBP to each well. Culture medium; NBP single-drug group add 100μL of culture medium containing different concentration gradients of NBP to each well; each drug has 3 multiple wells for each concentration, and corresponding blank wells (only medium) and normal wells (drug concentration) Is 0). After 72 hours of drug action, add MTT working solution (5mg/mL), 20μL per well; after 4 hours at 37℃, remove the supernatant, add 150μL of DMSO (analytical purity); Wipe clean, and detect the optical density (OD) at 550nm on the microplate reader.

Use the following formula to calculate the inhibition rate of cell growth:

Inhibition rate (%)=( _normal OD value-OD value _{dosing hole} )/( _normal OD value-OD value _{blank hole} )×100%

According to the inhibition rate of each concentration, SPSS19.0 was used to calculate the IC _{50 of the} drug's half inhibitory concentration.

The test was repeated 3 times, and the IC ₅₀ value was expressed as the mean ± SD of the 3 test results.

6. Results

6.1 The influence of NBP on the IC _{50 value of ab-PTX}

The results of the ab-PTX single-drug group showed that ab-PTX has effects on human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer cells (HT29), human lung cancer cells (of A549), in vitro proliferation of human melanoma (of A375) is significantly inhibited, which means in the range of IC ₅₀ 5-50nM. A specific concentration (30μM) of NBP was added to the combined administration. Compared with ab-PTX alone, the IC _{50 of} each cell in the combined administration group did not change significantly, and the range was still 5-50nM. This result suggests that NBP does not significantly affect the effect of ab-PTX in inhibiting tumor cell proliferation in vitro.

Table 12 shows _{the IC 50} values of ab-PTX single agent and the combined administration of ab-PTX and NBP to inhibit the proliferation of 6 tumor cells in vitro.

_{Table 12 The IC 50} value (nM) of ab-PTX single agent and ab-PTX combined with NBP to inhibit the proliferation of 6 tumor cells in vitro

6.2 The effect of NBP on tumor cell proliferation in vitro

NBP alone affects human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer cells (HT29), human lung cancer cells (A549), human melanoma (A375) has no obvious inhibitory effect on in vitro proliferation. The inhibitory rate of 30 μM NBP on the proliferation of 6 cell lines is shown in Table 13.

Table 13 Inhibition rate of 30μM NBP alone on 6 cell lines (%)

细胞种类Cell type	抑制率(％，30μMNBP)Inhibition rate (%, 30μMNBP)
MDA-MB-231MDA-MB-231	3.43±3.953.43±3.95
JIMT-1JIMT-1	7.39±13.237.39±13.23
SK-OV-3SK-OV-3	12.37±7.4112.37±7.41
HT29HT29	10.07±7.9410.07±7.94
A549A549	5.12±7.365.12±7.36
A375A375	10.92±0.5710.92±0.57

7. Conclusion

NBP does not affect ab-PTX on human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer cells (HT29), human lung cancer cells (A549), Inhibition of human melanoma cell (A375) proliferation in vitro; and, NBP alone on human breast cancer cells (MDA-MB-231, JIMT-1), human ovarian cancer cells (SK-OV-3), human colon cancer Cells (HT29), human lung cancer cells (A549), and human melanoma (A375) have no obvious inhibitory effect in vitro.

Example 6: In vivo efficacy study of butylphthalide on peripheral neuropathy induced by Bortezomib chemotherapy

1. Experimental animals

SD (Sprague Dawley) rats, male, 180-200g, 72 rats.

2. Drugs

Bortezomib (Bortezomib): batch number: 20191102, CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd., dissolved in normal saline containing 2% DMSO before use.

Butylphthalide: oral grade (10kg/bottle), batch number: 518180803, made by CSPC Enbipu Pharmaceutical Co., Ltd. Dilute with vegetable oil to the target concentration before use.

Duloxetine hydrochloride, batch number QRQYD-JN, TCI (Shanghai) Chemical Industry Development Co., Ltd., was prepared with 2% DMSO in physiological saline before use.

3. Test method

3.1 Model preparation and administration method

In this experiment, male SD rats were administered with a dose of 0.3 mg/kg of Bortezomib and a volume of 5 mL/kg. The models were made by intraperitoneal injection on the 0th, 2, 4, 6, 8, 10, and 13 days.

NBP uses three doses of 3, 10 and 30 mg/kg bid, and the first oral administration of NBP is started 1 day before Bortezomib model. During the period of Bortezomib administration model, the first daily dose of NBP is 1 hour before Bortezomib administration. For administration, the interval between the second administration of NBP and the first administration is ≥4h, the administration volume is 10mL/kg, and the administration is continuous for 21 days.

The normal control group (Normal) was given normal saline containing 2% DMSO at the same frequency and administration method as Bortezomib, and vegetable oil was given the same frequency and administration method as NBP. (See Figure 11 for model building and administration time, and see Table 14 for details of animal grouping, administration method and dosage).

Table 14 Animal grouping and dosage table

3.2 Grouping

According to the baseline value of the heat pain threshold and body weight of the rats, the rats with the baseline value in the range of 3 to 6s were selected, and the rats were divided into groups according to 3.1 items in Table 14 below.

3.3 Observation indicators

(1) Body weight: All animals were weighed once before the test, and weighed at a fixed time every day after the start of the administration.

(2) Thermal pain threshold: measured on

days

0, 7, 14, and 21.

Method: Using the IITC Life Science Electronic Von Frey Anesthesio meter, adjust the intensity of the light source to obtain a baseline thermal pain threshold of about 3 to 6 seconds. At this time, the light intensity is 58%, and the cutoff value is set to 10s. Place the rat in the plastic compartment on the glass plate, and turn on the light source to illuminate the center of the rat's foot. Record the time of reflex withdrawal of the rat, that is, the thermal pain threshold. The measurement is performed once for each rat's left and right feet, and the measurement is repeated three times with an interval of ≥15 min. The average of the 6 thermal pain thresholds for each rat is calculated.

(3) Mechanical stimulation pain threshold: Measured at -1, 6, 13, and 20 days.

4. Results

4.1 Impact on weight

On the 21st day of the test, compared with the normal control group, the weight of the rats in the model group was significantly reduced (P<0.05); compared with the model group, there was no significant change in the weight of the rats in the three doses of

NBP

3, 10 and 30 mg/kg bid , See Figure 12 for details.

4.2 Impact on thermal pain threshold

On the 21st day of the test, compared with the normal control group, the thermal pain threshold of the model group was significantly lower; compared with the model group, the

NBP

3, 10 and 30 mg/kg bid groups could significantly reverse the decrease of the thermal pain threshold of the model rats. See Figure 13 for details.

4.3 Influence on the pain threshold of mechanical stimulation

On the 20th day of the experiment, compared with the normal control group, the mechanical stimulation pain threshold of the model group was significantly lower; compared with the model group, the

NBP

10 and 30 mg/kg bid groups significantly reversed the decrease of the mechanical stimulation pain threshold of the model rats. See Figure 14 for details.

5 Conclusion

Under the conditions of this experiment, NBP can effectively relieve the symptoms of peripheral neuropathy in the rat CIPN model induced by Bortezomib in a dose-dependent manner.

Example 7: Test of the effect of NBP on the anti-tumor activity of Bortezomib in vitro

1. Cells and culture environment

Table 15 Cells and required medium

The above cell culture environment is 37°C and 5% CO ₂ .

2. Test purpose

To investigate whether NBP affects the effect of Bortezomib on inhibiting tumor cell proliferation in vitro.

3. Drugs

Bortezomib API, manufacturer: CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd., batch number: 20190802.

Butylphthalide, oral grade (10kg/bottle), manufacturer: CSPC Enbipu Pharmaceutical Co., Ltd., batch number: 518180803.

4. Experimental design

The test is divided into Bortezomib single-drug group, combined administration group (Bortezomib/NBP), and NBP single-drug group. Each group has a different drug concentration gradient.

Bortezomib single-drug group: ① MM.1S, Jeko-1, HT29 cells: Bortezomib starts at 20 nM, and there are 8 concentrations of 1.5× decreasing dilution, which are 20, 13.33, 8.89, 5.93, 3.95, 2.63, 1.76, 1.17nM; ②The final concentration of Bortezomib in A549 cells is 200nM as the initial concentration, and there are 8 concentrations in 2× decreasing dilution, respectively 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56nM; ③The final concentration of Bortezomib in DU145 cells is 100nM is the initial concentration, and 2×decreasing dilution sets a total of 8 concentrations, namely 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78nM.

Bortezomib/NBP group: The final concentration of NBP is 30μM, and the concentration gradient of Bortezomib is the same as the final concentration of Bortezomib single-drug group.

The action time of the drug was 72h.

5. Test method

Cells in the logarithmic growth phase of conventional culture were seeded in a 96-well plate in a certain amount, with 100 μL per well. For suspension cells MM.1S and Jeko-1, on the day of inoculation, in the Bortezomib group, add 100 μL of culture medium containing different concentration gradients of Bortezomib to each well, and add 100 μL of culture medium containing different concentration gradients of Bortezomib and 30 μM NBP to each well of the Bortezomib/NBP combined administration group. In the NBP single-drug group, 100μL of culture medium containing different concentration gradients of NBP was added to each well. For adherent cells HT29, A549 and DU145, the above-mentioned drugs were added 24 hours after adherence. There are 3 repeat holes for each drug concentration, and corresponding blank holes (only culture medium, no tumor cells inoculated) and normal holes (medium inoculated with tumor cells, drug concentration is 0). After 72 hours of drug action, add MTT working solution (5mg/mL), 20μL per well; after 4 hours at 37℃, remove the supernatant, add 150μL of DMSO (analytical purity); Wipe clean, and detect the optical density (OD) at 550nm on the microplate reader.

Use the following formula to calculate the inhibition rate of cell growth:

The test was repeated 3 times, and the IC ₅₀ value was expressed as the mean ± standard deviation of the 3 test results.

6. Results

The results showed that the Bortezomib single-drug group and the Bortezomib/NBP combined administration group had a significant inhibitory effect on the in vitro proliferation of MM.1S, Jeko-1, HT29, A549, and DU145, and the _{average IC 50} range was 1-20 nM. , Suggesting that NBP did not affect the effect of Bortezomib in inhibiting tumor cell proliferation in vitro. See Table 16 for details.

_{Table 16 The IC 50} value (nM) of Bortezomib and Bortezomib/NBP combined administration on 5 cell lines

NBP alone acts on human multiple myeloma cells (MM.1S), human mantle cell lymphoma cells (Jeko-1), human colon cancer cells (HT29), human lung cancer cells (A549), human prostate cancer cells (DU145) The in vitro proliferation has no obvious inhibitory effect. The inhibitory rate of 30μM NBP alone on the proliferation of 5 cell lines is shown in Table 17.

Table 17 Inhibition rate (%) of 30μM NBP alone on the proliferation of 5 cell lines

Conclusion: NBP does not affect Bortezomib on human multiple myeloma cells (MM.1S), human mantle cell lymphoma cells (Jeko-1), human colon cancer cells (HT29), human lung cancer cells (A549), and human prostate cancer cells. (DU145) Inhibition of proliferation in vitro.

Example 8: Test of the effect of NBP on the anti-tumor efficacy of Bortezomib in vivo

1. Test system

1.1 Experimental animals

NU/NU mice, female, 18-20g, 42 mice.

1.2 cell line

MM.1S cells (human multiple myeloma cells): purchased from Nanjing Kebai Biotechnology Co., Ltd.

2. Test purpose

In this experiment, the NU/NU mouse MM.1S cell transplanted tumor model was used to verify the effect of NBP on the anti-tumor efficacy and PK behavior of Bortezomib.

3. Drugs

Bortezomib API, manufacturer: CSPC Zhongqi Pharmaceutical Technology (Shijiazhuang) Co., Ltd., batch number: 20191102; dissolve in normal saline containing 2% DMSO before use.

Butylphthalide, oral grade (10kg/bottle), manufacturer: CSPC Enbipu Pharmaceutical Co., Ltd., batch number: 518180803; dilute with vegetable oil to the target concentration before use.

Palbociclib API, manufacturer: Shanghai Fangnan Biotechnology Co., Ltd., batch number: AL-0127-API-1811001; prepared with propylene glycol and 20% hydroxypropyl-β-cyclodextrin solution at a ratio of 5:95 before use Target concentration solution.

4. Test method

4.1 Model preparation

After resuscitating and passing MM.1S cells in vitro to a sufficient amount, after counting by microscope, dilute the cells with serum-free medium to adjust the number of cells to about 1×10 ⁸ cells/mL, and place the cell suspension in an ice water bath.

The MM.1S cell suspension was drawn with a sterile syringe to inoculate the subcutaneous tissues of the forelimb axilla of NU/NU mice. The inoculation volume was 0.1 mL per mouse, containing about 1.0×10 ⁷ tumor cells to prepare NU/NU mouse MM.1S transplantation. Tumor model.

4.2 Method of administration

Bortezomib single-drug group: intraperitoneal injection twice a week (on the 0, 3, 7, 10, 15 days, once a day), 0.5 mg/kg;

NBP single-drug group: oral gavage twice a day (administration started on day 0), 60 mg/kg, the interval between the second administration of NBP and the first administration ≥ 4h;

Palbociclib single-drug group: once a day by intragastric administration (dose on day 0), 70mg/kg;

NBP/Bortezomib combined administration group: The frequency and dose of administration are the same as those of the NBP and Bortezomib single-drug groups.

Bortezomib/Palbociclib combined administration group: The frequency and dose of administration are the same as those of the Bortezomib and Palbociclib single-drug groups.

NBP/Bortezomib/Palbociclib combined administration group: The frequency and dose of administration are the same as those of the NBP, Bortezomib and Palbociclib single-drug groups.

The solvent group was given solvents at the same frequency and administration method as Bortezomib and NBP.

The dosing cycle is 16 days, and the dosing volume is 10 mL/kg.

4.3 Observation indicators and evaluation indicators

4.3.1 Observation indicators

4.3.2 Tumor volume evaluation

After the animals were grouped, the tumor length and short diameter were measured twice a week.

(1) Tumor volume: V=1/2×A×B ²

(2) Relative tumor volume:

(3) Relative tumor volume growth rate:

(4) Tumor inhibition rate:

Note: V: tumor volume; A: tumor length; B: tumor width; RTV: relative tumor volume; TVnd: tumor volume on day n; TV0d: tumor volume on day 0; RTVxnd: average relative tumor volume on day n; TV _X n: average tumor volume on day n of the drug-administered group; TV _X0 : average tumor volume on day 0 of the drug-administered group; TV _M n: average tumor volume on day n of the solvent group; TV _M0 : average tumor volume on the 0th day of the solvent group

4.3.3 Tumor weight

4.3.4 PK blood sampling and testing

Blood was collected before the last dose of Bortezomib (0h), 5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h after the administration (see Table 18 for details), the mice in the solvent group were only collected at 0h, heparin was anticoagulated, and centrifuged. The plasma was stored at -80℃ for later use. The protein precipitation-LC/MS/MS method was used to determine Bortezomib and butylphthalide in the plasma.

Table 18 Blood collection schedule of mice in each group

组别Group	动物编号Animal number	采血时间点Blood sampling time point

溶剂组Solvent group	1～61～6	0h0h
各给药组Each administration group	1～61～6	0，5min，15min，30min，1h，2h，4h，8h，24h0, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 24h

5. Test results

5.1 Impact on weight

From the body weight data, there was no significant difference in the body weight of the mice in each group (P>0.05), indicating that Bortezomib, Palbociclib and NBP had no effect on the body weight of the mice. See Figure 15 for details.

5.2 Effect on tumor volume

At the end of the test, compared with the Bortezomib single-agent group, NBP/Bortezomib combined administration did not have a significant negative effect on the antitumor efficacy of Bortezomib; compared with the Bortezomib/Palbociclib combined administration group, the NBP/Bortezomib/Palbociclib combined administration group In the administration group, NBP also did not have a significant negative effect on the anti-tumor efficacy of the combined administration of Bortezomib/Palbociclib. The tumor volume of each group is shown in Figure 16, and the weight, TV, RTV, TGI%, T/C%, tumor weight, etc. of the test endpoint (day 14) are shown in Table 19.

Table 19 Summary table of various index parameters of NU/NU mice on the 14th day of the test (mean±SD, n=6)

Note: Bor stands for Bortezomib and Pal stands for Palbociclib.

Conclusion: In this trial, NBP has no significant effect on the anti-tumor efficacy of Bortezomib and Bortezomib/Palbociclib combined administration.

5.3 Impact on PK

The results showed that the Bortezomib exposure in the Bortezomib group, the NBP/Bortezomib group, the Bortezomib/Palbociclib group and the NBP/Bortezomib/Palbociclib group was basically the same, suggesting the in vivo pharmacokinetics of NBP on Bortezomib single agent and Bortezomib/Palbociclib combined administration None of the behaviors had a significant impact. The main pharmacokinetic parameters are shown in Table 20, Table 21 and Table 22.

Table 20 Summary table of main pharmacokinetic parameters of Bortezomib in NU/NU mouse plasma (mean±SD, n=6)

Table 21 Summary table of main pharmacokinetic parameters of NBP in plasma of NU/NU mice (mean±SD, n=6)

Note: In Tables 20 and 21, Bor stands for Bortezomib, Pal stands for Palbociclib, C _max stands for maximum plasma concentration, AUC _0-t stands for the area under the drug-time curve from 0 to t, t _1/2 stands for half-life, and Vd stands for Apparent volume of distribution, Cl represents the clearance rate, MRT _0-t represents the average residence time from 0 to t.

Table 22 Comparison results of the average plasma exposure of Bortezomib and NBP in the combination group and the single-drug group

Note: Bor stands for Bortezomib and Pal stands for Palbociclib.

_{The ratios of C max} and AUC _{0-t of} Bortezomib in the combined administration group of Bortezomib/Palbociclib, NBP/Bortezomib/Palbociclib and NBP/Bortezomib group to Bortezomib single agent group were 1.01-1.13 and 0.81-1.11, respectively. C _max and AUC _0-t after logarithmic conversion, the ratios of the combined administration of Bortezomib/Palbociclib, NBP/Bortezomib/Palbociclib and NBP/Bortezomib group to the Bortezomib single-drug group were 1.00～1.05 (LNC _max ) and 0.96～1.02, respectively (LNAUC _0-t ), so the in vivo exposures of Bortezomib single-drug and combined administration groups are basically the same.

_{The ratios of C max} and AUC _{0-t of} NBP in the NBP/Bortezomib/Palbociclib and NBP/Bortezomib groups to the NBP single-drug group were 1.08-1.24 and 1.03-1.83, respectively. After logarithmic transformation of C _max and AUC _0-t , the ratios of the combined NBP/Bortezomib/Palbociclib and NBP/Bortezomib groups to the NBP single-drug group were 1.02～1.05 (LNC _max ) and 1.00～1.11 (LNAUC _{0- t} ). Due to the obvious individual differences in mice after intragastric administration of NBP, the number of mice in each test group is small. The ratio of exposure levels between the combined administration group and the single-drug group after logarithmic transformation shows that NBP single-drug and There was no significant difference in the in vivo exposure of the combined administration group.

Conclusion: In this experiment, NBP and Bortezomib basically did not affect each other's pharmacokinetic behavior in mice.

Claims

Application of butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product in the preparation of drugs for preventing, relieving or treating peripheral neuropathy.
The application according to claim 1, wherein the peripheral neuropathy is preferably drug-induced peripheral neuropathy, and more preferably chemotherapeutic drug-induced peripheral neuropathy.
The application according to any one of claims 1-2, wherein the prevention, alleviation or treatment of peripheral neuropathy includes but is not limited to prevention, alleviation or treatment of paresthesia and movement disorders caused by chemotherapy drugs, such as pain , Heat sensory abnormalities, impaired motor coordination, etc.
The application according to any one of claims 1-3, wherein the drug contains a therapeutically effective amount of butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opened products or The salt of the ring-opening product.
The application according to any one of claims 1 to 4, wherein the medicine is prepared into clinically accepted preparations, such as oral preparations, injection preparations, topical preparations, topical preparations, etc., preferably oral preparations and injections preparation.
The application according to any one of claims 1 to 5, wherein the medicine is in a single-dose dosage form or a divided-dose dosage form.
The application according to any one of claims 1-6, wherein the drug is an oral preparation; the oral preparation contains butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, The ring-opening product or the salt of the ring-opening product is about 1 mg to about 1000 mg, preferably about 1 mg to about 500 mg, or about 1 mg to 300 mg, or about 1 mg to 200 mg, or about 5-180 mg, or about 10-150 mg, or about 30-120 mg, or about 50-120 mg, or about 80-120 mg, or about 90-110 mg, or about 100 mg, based on butylphthalide.
The application according to claim 7, wherein the daily dosage of the oral preparation is about 1 mg to about 10000 mg, preferably about 10 mg to about 5000 mg, or about 20 mg to 3000 mg, or about 30 mg to 2000 mg, Or about 50mg-1500mg, or about 70mg-1200mg, or about 100mg-1000mg, or about 200mg-900mg, or about 300mg-800mg, or about 400mg-700mg, or about 500mg-600mg, or about 60mg-800mg, or about 60mg-600mg, or about 100mg-800mg, or about 100mg-600mg, or about 200mg-600mg, or about 200mg-800mg, or about 300mg-600mg, or about 400mg-600mg, or about 400mg-800mg, as butylbenzene Phthal meter.
The application according to claim 8, wherein the oral preparation is administered once a day, and butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, ring-opening products or The oral preparation of the salt of the ring-opening product is about 60-800 mg, for example, about 60 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, or 800 mg of the oral preparation per administration, in terms of butylphthalide; or daily administration Two doses of medicine, each time the oral preparation of butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product is administered about 30-400 mg, such as oral preparations each time About 30mg, 50mg, 100mg, 150mg, 200mg, 250mg, 300mg, 350mg or 400mg, calculated as butylphthalide; or, administered 3 times a day, each time butylphthalide or its optical isomers or prodrugs Oral preparations of, deuterated substances, metabolites, ring-opening products or salts of ring-opening products are about 20-300 mg, such as about 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, or 300 mg per administration of oral preparations, calculated as butylphthalide .
The application according to any one of claims 1 to 6, wherein the drug is an injection preparation; the injection preparation contains butylphthalide or its optical isomer, prodrug, deuterium, metabolite, The ring-opening product or the salt of the ring-opening product is about 0.001mg/ml-100mg/ml, preferably, about 0.005mg/ml-50mg/ml, or about 0.01mg/ml-10mg/ml, or about 0.1mg/ ml-5mg/ml, or about 0.1mg/ml-3mg/ml, or about 0.1mg/ml-1mg/ml, or about 0.12mg/ml-0.80mg/ml, or about 0.15mg/ml-0.50mg/ ml, more preferably about 0.20mg/ml-0.40mg/ml, or about 0.20mg/ml-0.30mg/ml, or about 0.25mg/ml, in terms of butylphthalide.
The use according to claim 10, wherein the daily dosage of the injection preparation is about 1 mg to about 1000 mg, preferably about 5 mg to about 500 mg, or about 10 mg to 300 mg, or about 15 mg to 200 mg, Or about 20mg-150mg, or about 25mg-120mg, or about 30mg-100mg, or about 35mg-90mg, or about 40mg-80mg, or about 45mg-70mg, or about 50mg-60mg, or about 1mg-100mg, or about 2mg-80mg, or about 5mg-75mg, or about 10mg-50mg, or about 15mg-50mg, or about 20mg-50mg, or about 25mg-75mg, or about 25mg-50mg, in terms of butylphthalide.
The application according to claim 11, wherein the injection preparation is administered once a day, and butylphthalide or its optical isomers, prodrugs, deuterated products, metabolites, and ring-opening products are administered each time Or the injection preparation of the salt of the ring-opening product is about 1 mg to about 100 mg, preferably about 1 mg, 2 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50 mg, 55 mg, 60 mg, 65 mg, 70mg, 75mg, 80mg, 85mg, 90mg, 95mg or 100mg, as butylphthalide; or twice a day, butylphthalide or its optical isomers, prodrugs, deuterated substances, metabolism The injection preparation of the salt of the product, the ring-opening product or the ring-opening product is about 1 mg to about 50 mg, preferably about 1 mg, 2 mg, 2.5 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, 50mg.
The application according to any one of claims 1-12, wherein the chemotherapeutic drugs include but are not limited to one or more of the following drugs: (1) chemotherapeutic drugs that act on the microtubule system or anti-mitosis , Including: taxane drugs, such as paclitaxel, docetaxel, etc.; or vincristine drugs, such as vincristine or vinblastine, etc.; or epothilone drugs, such as ixabepilone; Or protease inhibitor drugs, such as bortezomib, etc.; or (2) chemotherapeutic drugs that interfere with DNA synthesis, including platinum drugs, such as cisplatin, carboplatin, or oxaliplatin; or (3) immunomodulators Drugs, such as thalidomide, lenalidomide, etc.
The application according to any one of claims 1-13, wherein the butylphthalide or its optical isomer, prodrug, deuterium, metabolite, ring-opening product or salt of the ring-opening product can be It is used in combination with one or more of other peripheral neuropathy treatment drugs to prepare the drug; preferably, the other peripheral neuropathy treatment drug is duloxetine or its salt, monosialotetrahexose ganglion Sodium Glucoside.